The proposed experiments would utilize two unique strains of Long-Evans rats, produced by genetic selection, with very different red cell 2,3-diphosphoglycerate (DPG) levels and 7 mm Hg differences in whole blood p50 (partial pressure at which Hemoglobin (Hb) is half saturated). This DPG difference is due to the effects of two alleles at one locus. DPG genotype is strongly associated with genotype at the III-beta globin locus. Similar associations exist in the mouse and possibly in man. It has been unclear whether there is only the globin locus or whether there are two linked loci with great linkage disequilibrium. Recent phosphofructokinase (PFK) kinetic data from this laboratory strongly support the two locus hypothesis as does the existence of one recombinant which has now been progeny tested. We propose to develop a DPG locus genotyping assay based on PFK inhibition differences and use it to confirm recombinants and test for linkage disequilibrium in 22 inbred rat strains. Oxygen equilibria studies will test for functional differences between the III-beta containing Hbs. Studies of PFK isozyme and subunit structure will hopefully reveal which PFK subunit differs between rat strains and which tissues share the difference. If no recombinants are found, the alternative mechanisms for the association of chromosomal inversion and posttranslational modification will be investigated. These experiments are designed to prove the existence of two linked loci and provide important data on the functional differences between the gene products in an attempt to understand the mechanism of this great linkage disequilibrium.